Inheritance of DNA methylation in Coprinus cinereus.

Abstract

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.Postreplicative methylation of DNA cytosine residues is a common feature of procaryotic and eucaryotic DNAs. In eucaryotes, cytosine methylation is believed to be a controlling mechanism in gene expression (for examples, see references 5 and 14) and development (7,8). Since most methylation in animals and fungi is at the nucleotide doublet CpG (2, 17), the isoschizomer pair of restriction endonucleases, HpaII and MspI, has provided a powerful tool for the study of the methylation patterns of specific DNA sequences (3, 18). These two enzymes recognize the same sequence (5'-CCGG-3'), but HpaII is sensitive to CpG methylation, while MspI is sensitive to CpC methylation (9).Coprinus cinereus is a particularly useful organism for the study of eucaryotic DNA methylation. This basidiomycete has a small nuclear genome size of 38,000 kilobase pairs (kb) (6), which facilitates molecular analyses. In addition, genetic studies and experimental manipulation of all phases of its life cycle are straightforward and simple. We have shown previously that the nuclear genome of this fungus is extensively methylated at the nucleotide doublet CpG (M. E. Zolan and P. J. Pukkila, in Molecular Genetics of Filamentous Fungi, in press). In that study, we also examined the inheritance of DNA methylation at a centromere-linked locus, termed 16-1, which is methylated in one geographical isolate (Okayama-7) but not in others. Tetrad analysis of progeny from a cross between Okayama-7 and PJP52 (which is unmethylated at 16-1) revealed that methylated tracts are inherited 2:2 during meiosis. That is, progeny that received the chromosome containing locus 16-1 from Okayama-7 were always methylated at that locus, and progeny that inherited locus 16-1 from PJP52 were not methylated at that locus. However, we observed altered lengths of methylated tracts among the methylated progeny. In this study, we asked whether these alterations are a direct consequence of the meiotic process, and we also asked whether the altered tracts resulting from de novo methylation are transmitted as simple Mendelian alleles.MATERIALS AND METHODS C. cinereus strains and growth. The two parents, Okayama-7 and PJP52, of MZ1, the first cross in this study, were described previously (4). The remainder of the strains used were progeny of MZ1 and are described in al. (4). To isolate oidia, 5 ml of sterile H20 was poured onto the surface of a confluent plate of a monokaryotic cultu...

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“…(b) DNA sequencing Genomic DNA was extracted from lyophilized mushrooms or vegetative mycelia using CTAB (hexadecyltrimethyl-ammonium bromide) in a standardized DNA extraction protocol (Zolan & Pukkila 1986). Genomic DNA was used as the template for PCR amplification of the first 900 base pairs of the large subunit of the nuclear ribosomal DNA gene.…”

Fruiting body development in Coprinus cinereus: regulated expression of two galectins secreted by a non-classical pathway The GenBank accession number for the sequence reported in this paper is AF130360

“…Determination of mRNA levels by RT-PCR. Total RNA was isolated from powdered, lyophilized Coprinus tissues by a guanidinium isothiocyanate procedure (Chomczynski & Sacchi, 1987) and genomic DNA of homokaryon AmutBmut was obtained by the method of Zolan & Pukkila (1986). The cDNA templates were generated using a DNase I and cDNA synthesis kit (Gibco-BRL) starting with 2 µg total RNA and an oligo-poly(dT) "& primer.…”

Molecular fingerprinting of Fusarium oxysporum f. sp. passiflorae isolates using AFLP markers

“…The extraction of DNA from the isolates was performed using the CTAB method ((C 16 H 33 )N(CH 3 ) 3 Br, cetyltrimethylammonium bromide hexadecyltrimethylammonium bromide) described by Zolan and Pukilla (1986). DNA quantification was performed using visual comparison to the intensities of fragments of a series of known concentrations of Lambda DNA (Invitrogen), after electrophoresis (3 v cm -1 ) in 1.0 % agarose gels containing ethidium bromide (1.0 µg mL -1 ).…”